DESCRIPTION: The proposed project examines synaptic vesicle processing at the vertebrate nerve-muscle synapse. Because neurotransmitter-containing vesicles are dynamically recycled within the presynaptic terminal during activity, vesicle processing influences the amount of transmitter released, and hence the strength of the synapse. The project's aim is to characterize key processing steps and thereby determine which might be amenable to pharmacological intervention in diseases, such as Parkinson's, depression and Eaton-Lambert's, where alteration of synaptic strength is the primary pathology. Processing steps considered include endocytosis, sorting of membrane proteins, and selection of transmitter release sites. Experiments make use of a unique thin nerve-muscle preparation which is highly stereotyped and accessible, allowing electrophysiological, light microscopic, and electron microscopic evidence to be compared, all at the level of individual terminal boutons. A new technique is introduced. It allows brief pulses of externally-applied endocytic probes to be internalized and subsequently followed, thereby labeling specific processing pathways in boutons for both light- and electron-level imaging. This technique will be combined with direct measurement of transmitter release while the synapse is experimentally manipulated. Thus vesicle processing will be related to synaptic strength under a variety of experimental conditions.